Dynamic reorganization of the actin cytoskeleton is the basis for cell morphology and migration. In different cellular locales F-actin accessory proteins undergo transient Ser/Thr phosphorylation and act as molecular transducers, switching on/off to control F-actin. Multiple upstream kinases phosphorylate these accessory proteins in response to signals. The opposing phosphatases are complexes of PP1 catalytic and regulatory subunits physically targeted and under stringent negative control. Phosphorylation of PP1 subunits and phosphoinhibitor proteins couple the activity of kinases with PP1 complexes, producing reciprocal changes that generate the on/off switching behavior. The goal of this Project is to understand how signaling pathways control dynamic reorganization of the cytoskeleton, through regulation of F-actin accessory proteins by specialized forms of protein Ser/Thr phosphatase PPI. this Project will study two different PP1 complexes. One complex is PP1C8 plus the regulatory-targeting subunit called MYPT1 (myosin phosphatase targeting subunit), which binds to actomyosin-II in stress fibers and uses ankryin-repeats to bind the F-actin-anchoring proteins ezrin/radixin/moesin (ERM), and the actin-capping protein adducin. MYPT1 phosphorylation regulates the PP1 activity, but the consequences on the F-actin cytoskeleton have not been examined. The other PP1 complex, discovered by this project, is composed of PP1Ccalpha plus tensin, an F-actin anchoring phosphoprotein concentrated in mature focal adhesions. Specific Aim 1 will test the hypothesis that the activity of MYPT1-PP1C toward accessory proteins such as ERM proteins and adducin alters cortical F-actin dynamics. Wild type MYPT1 and MYPT1 mutated in its regulatory phosphorylation sites will be over-expressed with PP1C8 as dominant active and inactive phosphatases. Alternatively, endogenous MYPT1 will be depleted by siRNA silencing. The phosphorylation of ERM proteins and adducin will be monitored in parallel with changes in F-actin distribution, cell morphology and cell migration. Specific Aim 2 will test the hypothesis that the C terminal PTB domain in tensin directly binds PP1Ccalpha and functions as a regulatory-targeting subunit. The tensin binding of PP1C is proposed to be in competition with P-Tyr ligands, modulating recruitment of PP1 in time and space. Experiments will test whether the tensin.PP1C complex serves to reduce Ser/Thr phosphorylation in specific substrates and thereby alter turnover of fibrillar adhesions and modify cell motility. This Project connects signaling pathways to cortical F-actin and focal adhesions that are critical to cell polarity, and cell migration, which requires PI3K-dependent formation of polyphosphoinositides. Our experimental plan involves use of microscopic imaging and depends on collaborative interactions with other Projects.